One of the most critical steps in the progression to malignancy is the acquisition by tumor cells of the ability to metastasize. The hypothesis to be tested in this proposal is that downregulation of Rap1GAP contributes to thyroid tumor progression. This notion is based on data showing that Rap1GAP is highly expressed in normal human thyroid cells, and that its expression is dramatically reduced in the majority of invasive papillary thyroid carcinomas. Studies in human thyroid carcinoma cell lines revealed a striking correlation between loss of Rap1GAP and loss of epithelial structure. Rap1GAP-deficient tumor cells lacked E-cadherin and acquired the expression of vimentin, indicative of epithelial-to-mesenchymal transition. These cells exhibited enhanced migratory and invasive properties compared to tumor cells that retained Rap1GAP. Restoring Rap1GAP to Rap1GAP-deficient cells inhibited cell migration, invasion and anchorage-independent proliferation. Silencing Rap1GAP expression in thyroid carcinoma cells that retain an epithelial morphology caused the cells to disperse, enhanced the dissociation of cell aggregates, and dysregulated E-cadherin, suggestive of defects in cell/cell adhesion. As disruption of cell/cell junctions is associated with serious pathological consequences, we propose that downregulation of Rap1GAP contributes to the pathogenesis of thyroid tumors by attenuating cell/cell adhesion. In vitro studies will explore the molecular mechanism through which decreased Rap1GAP expression attenuates cell/cell adhesion and assess whether eliminating Rap1GAP endows tumor cells with altered migratory properties and invasive potential. As TSH regulation of Rap1GAP is lost from the human thyroid tumor cell lines, but is likely to be retained in primary thyroid tumors, complementary studies will be performed in differentiated rat thyroid cells. These studies will be conducted in cells in three-dimensional environments, conditions that more closely reproduce the matrix compliance of human tissues and where cell/cell contacts are maximized. The in vitro studies are complemented with studies in human tumor specimens that will identify the subtypes of thyroid tumors in which Rap1GAP expression is decreased and probe the clinical significance of loss of Rap1GAP. In summary, this proposal presents a highly cohesive plan to investigate the contribution and clinical significance of Rap1GAP depletion to the progression of human thyroid tumors.